B CELL RECEPTOR SIGNALING IN RECEPTOR EDITING AND LEUKEMIA A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY LAURA BAILEY RAMSEY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY MICHAEL A. FARRAR, ADVISER JULY 2009 © Laura Bailey Ramsey 2009 i Acknowledgements I would like to thank my family and friends for all their support, for without it I would not have accomplished all that I have. I would especially like to thank my husband Colin, for supporting me through the tough times and celebrating with me during the happy times. I am eternally grateful to my parents and grandparents for their support and unconditional love. These last five years would not have been the same without the support of my “Minnesota Mom,” Lyn Glenn. They have all encouraged me to accomplish my dreams, and without them I would not have been able to do it. I would like to thank the Behrens lab for all their support and patience with me when I started graduate school. They introduced me to the world of immunology and B cells, and for that I am forever grateful. Tim Behrens was a great mentor and project director for the first two years of my graduate career. Brian Schram and Keli Hippen were especially helpful in teaching me all about B cells. Jason Bauer, Thearith Koeuth, and Emily Gillespie have taught me many things about microarrays and continue to support me at the University of Minnesota. Jessica Oehrlein and Amanda Vegoe took wonderful care of my mice. Ward Ortmann and Karl Espe provided much needed technical support. The Farrar lab welcomed me warmly after Tim’s departure. I am especially grateful to Mike for learning a new project and providing good advice and support. Lynn Heltemes-Harris and Mark Willette provided much of the data for Chapter 4, and without their hard work dissecting tumor mice this thesis would be incomplete. Linxi Li and Casey Katerndahl provided helpful discussions as fellow members of the B cell group in the lab. Matt Burchill provided support and advice on this thesis even after leaving the lab. I would like to thank Jianying Yang, Bao Vang, and Shawn Mahmud for helpful discussions, despite the fact that they are “T cell people.” My mice were well taken care of by Amanda Vegoe, Rachel Agneberg, Christine Andersen and Josh Bednar. I am grateful to everyone in the Farrar lab for their friendship and support. I would like to thank other members of the Center for Immunology for their support and advice. James McLachlan provided excellent advice on this thesis and post-doctoral interview preparation. Colleen Winstead provided moral support in the library during the writing of this thesis. Paul Champoux and Nisha Shah provided cell sorting support and flow cytometry expertise. I am grateful to my committee for their guidance and support. The Molecular, Cellular, Developmental Biology & Genetics staff provided support throughout my graduate career, especially during the transition period from one lab to another. I am also grateful to the graduate school for funding through the Doctoral Dissertation Fellowship. I am thankful for my fellow graduate students in the MCDB&G program for moral support and many wonderful friendships that will undoubtedly last a lifetime. They have made my graduate career enjoyable and provided many memorable experiences. ii Dedication This dissertation is dedicated to everyone who has supported me during the past five years while I attended graduate school. I love you all! iii Abstract There are many unanswered questions in the B cell field, but one of paramount importance is how B cells are tolerized to avoid autoimmunity. The majority of developing B cells are probably self-reactive, and many that make it through B cell development to the periphery show evidence of receptor editing. The question remains as to how the B cells signal to re-induce the editing machinery, whether it is a positive reactivation signal or the release of an inhibitory tonic signal. We propose that it is the release of a tonic signal through the B cell receptor that inhibits recombination when the BCR is present on the surface, but when the BCR binds self-antigen it is internalized, the inhibitory signal is abrogated, and the recombination machinery is reactivated. We tested this hypothesis using several transgenic models, including RAG2-GFP mice, HEL Ig mice, and anti-κ mice. We also used several mice deficient in BCR signaling to test this hypothesis, and found that reduced BCR signaling induces more receptor editing. When we tested mice with excessive BCR signaling or pharmacologically mimicked the BCR signal, we found a decrease in receptor editing. These experiments provide compelling evidence for the inhibitory tonic signaling hypothesis and against the activation signaling hypothesis. Another question unanswered in the B cell field is how B cells are transformed into leukemia and lymphoma. There have been whole genome analysis studies to show that genes involved in the BCR signaling pathway are involved in many B cell malignancies, including leukemia, lymphoma and myeloma. Our studies in mice provide evidence that in combination with constitutive STAT5 activation, a loss of genes involved in B cell development (ebf1 and pax5) or pre-BCR signaling (blnk, iv PKCβ, and btk) results in leukemia. These are genes that have been shown to be deleted in B cell acute lymphoblastic leukemia (ALL). We also demonstrate an increase the phospho-STAT5 levels in adult BCR-ABL+ ALL patients. Herein we have developed a new mouse model for B-ALL that may be useful in testing and perfecting treatment protocols used on ALL patients. v Table of Contents List of tables vi List of figures vii Author/publication information ix List of abbreviations x Chapter 1. Introduction 1 B cell Development 1 Transcriptional Control of B cell Development 3 Interleukin-7 Receptor Signaling 5 V(D)J Recombination 7 Pre-BCR Signaling 9 BCR Signaling 13 Receptor Editing 18 B cell Acute Lymphoblastic Leukemia 22 Objectives 26 Chapter 2. BCR Basal Signaling Regulates Antigen-Induced Ig Light Chain 36 Rearrangements Chapter 3. Tonic BCR Signaling Represses Receptor Editing via the Ras 87 and Calcium Signaling Pathways Chapter 4. STAT5 Cooperates with Defects in B Cell Development to Initiate 113 Progenitor B Cell Leukemia Chapter 5. Discussion 166 References 172 Appendix: Permission letter from the Journal of Immunology 187 vi List of Tables Chapter 2. Table 1. Selected differentially expressed transcripts in Ag-treated 85 immature B cells. Chapter 4. Table I. The top 50 probe sets by fold change (Ia) or p-value (Ib) 163 vii List of Figures Chapter 1. Figure 1. B cell development 28 Figure 2. Transcription Factor Network Involved in B cell Development 30 Figure 3. B cell Receptor Signaling Pathway 32 Figure 4. V(D)J Recombination & LC Receptor Editing 34 Chapter 2. Figure 1. Xid Immature B cells show impaired antigen-induced 69 activation responses, but normal IgM downregulation Figure 2. RAG2-GFP responses in xid and lynnull immature B cells 71 Figure 3. Relationship between RAG2-GFP expression and surface IgM 73 Figure 4. Back-differentiation of antigen-stimulated immature B cells 75 Figure 5. Expression of immature B cell markers following IL-7 77 bone marrow culture Figure 6. Antigen-treated immature B cells show similar gene 79 expression profiles as cells that have lost the BCR or have been treated with a tyrosine kinase inhibitor Figure 7. Suppression of RAG2-GFP and Ig rearrangements in 81 immature B cells by PMA and Ionomycin Figure 8. Pre-B cell LC editing to membrane self-antigen is inhibited 83 by PMA/Ionomycin viii Chapter 3 Figure 1. Light chain expression in BM chimeras 107 Figure 2. Editing ratio in BM chimeras 109 Figure 3. Model of Receptor Editing Inhibition by BCR Signals 111 Chapter 4 Figure 1. Spontaneous tumors in STAT5b-CA mice 132 Figure 2. Cyclin D2 and myc expression in STAT5b-CA ALL 134 Figure 3. STAT5b-CA causes a significant increase in tumor incidence 136 in all mouse crosses Figure 4. Flow cytometric analysis of leukemic mice 138 Figure 5. CD79a expression 140 Figure 6. Microarray analysis of tumor samples 142 Figure 7. Canonical pathways differentially regulated in tumor samples 144 Figure 8. Expression of BCR signaling pathway genes 148 Figure 9. TNFR expression 151 Figure 10. Regulators of NFkB2 signaling 153 Figure 11. Cooperation response genes in STAT5b-CA x xid tumors 155 Figure 12. Total STAT5 protein expression 157 Figure 13. STAT5 activation in human ALL 159 Figure 14. STAT5 phosphorylation in different cytogenetic subsets of ALL 161 ix Author/Publication Information Chapter 2. BCR Basal Signaling Regulates Antigen-Induced Ig Light Chain Rearrangements Published in the April 1, 2008 issue of the Journal of Immunology (J Immunol. 2008 Apr 1;180(7):4728-41). I wrote much of the manuscript, re-analyzed data and prepared several of the figures, submitted the manuscript, responded to reviewers’ comments and made necessary revisions. Brian Schram and Lina Tze performed experiments, analyzed data, prepared figures and assisted in writing the manuscript. Jiabin Liu, Lydia Najera, Amanda Vegoe, and Keli Hippen assisted with experiments and provided discussion related to the manuscript. Richard R. Hardy provided samples for microarray analysis. Timothy Behrens and Michael Farrar co-directed the project. Chapter 3. Tonic BCR Signaling Represses Receptor Editing via the Ras and Calcium Signaling Pathways This manuscript is in preparation for submission. I performed the experiments, analyzed the data, prepared the figures, and wrote the manuscript. Amanda Vegoe helped dissect the mice and oversaw the mouse husbandry. Timothy Behrens conceptualized the project, which was directed by Michael Farrar.